ABSTRACT The CLC channels and transporters mediate anion transport through biological membranes. Genes encoding for CLC proteins are found in nearly all organisms, from bacteria to plants and humans. Mutations altering the properties of five of the nine human genes encoding for CLC homologues result in genetic disorders of bone, kidney, brain and muscle, highlighting the fundamental role of these proteins in a wide variety of tissues and cellular compartments. Despite their pathophysiological importance, our understanding of how these proteins function lagged far behind many other classes of ion channels and exchangers. This limits our ability to interpret their function in human physiology and to design targeted pharmacological interventions that would selectively manipulate their activity. Thus, our long-term goal is to elucidate the atomic basis for CLC Cl- channel and transporter function. Our proposal is articulated in three specific aims, each of which addresses a fundamental unanswered mechanistic question on CLC function. Our innovative use of synergistic experimental and computational approaches enables the formulation of specific hypotheses and their rigorous testing. In the first Aim we will determine the bases of substrate selectivity in the CLC Cl- channels. While selectivity of cation channels is well understood, nearly nothing is known on anion selectivity. We will probe the role of the protein backbone in this process using atomic-scale mutagenesis and will determine the consequences of these manipulations through structural, electrophysiological and computational experiments. Our second aim is to elucidate the coupling mechanism in the CLC exchangers. The CLC transporters exchange 2 Cl- for 1 H+ across biological membranes. Several disease-causing mutations affect this process through unknown mechanisms. Our goal is to elucidate the basis for Cl-/H+ coupling in the CLCs. We will utilize computational tools in conjunction to conventional and atomic mutagenesis to probe the dynamic rearrangements undergone by the protein to enable the formation of a pathway for H+ that is physically distinct from the route taken by the Cl- ions. The third aim is to determine the molecular origin of the functional divergence of the CLC channels from the transporters. Despite the availability of high resolution structural information for both subtypes, the molecular origin of this functional divergence remains unknown. We will use statistical phylogenetics and evolutionary bioinformatics to identify the most likely evolutionary sequence of events leading to the functional divergence. We will then functionally characterize sequences recapitulating these key evolutionary steps and use this information to identify a subset of amino acid substitutions necessary to enact the functional switch. Ultimately, these efforts will lead to new molecular and conceptual framework for the understanding of CLC function, which will enable the design of approaches for the amelioration of the disease conditions caused by the dysfunction of these proteins.